Glycoside Hydrolase Family 23

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• Author: ^^^Anthony Clarke^^^
• Responsible Curator: ^^^Anthony Clarke^^^

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Glycoside Hydrolase Family GHnn
Clan	GH-x
Mechanism	retaining/inverting
Active site residues	known
CAZy DB link
http://www.cazy.org/fam/GHnn.html

Substrate specificities

The glycoside hydrolases of this family are lytic transglyosylases (also referred to as peptidoglycan lyases) of both bacterial and bacteriophage origin, and family G lysozymes (EC 3.2.1.17; muramidase, peptidoglycan N-acetylmuramoylhydrolase, 1,4-β-N-acetylmuramidase, N-acetylmuramoylhydrolase) of eukaryotic origin. Both of these enzymes are active on peptidoglycan, but only the lysozymes are active on chitin and chitooligosaccharides. No other activities have been observed.

Kinetics and Mechanism

The enzymes of this family cleave the β-1,4 linkage between N-acetylmuramoyl and N-acetylglucosaminyl residues in peptidoglycan (Figure 1) . Only the lysozymes of this family are capable of releasing N-acetyl-d-glucosamine residues from chitodextrins, and neither catalyze (inter) transglycosylation reactions. The mechanism of the family G lysozymes has not been determined experimentally, but theoretical considerations based on crystallographic data [1] and modeling studies [2] suggest that they are inverting enzymes. On the other hand, the lytic transglycosidases, strictly speaking, are retaining enzymes. However, unlike lysozyme, they are not hydrolases but rather catalyse an intramolecular glycosyl transferase reaction onto the C-6 hydroxyl group of the muramoyl residue leading to the generation of a terminal 1,6-anhdyromuramoyl product thus lacking a reducing end [3]. The lytic transglycosylases require the peptide side chains in peptidoglycan for activity, accounting for their inactivity against chitin or chitooligosaccharides [4]. No detailed analyses involving both steady state and pre-steady state kinetic studies have been reported.

Catalytic Residues

Unlike most other glycoside hydrolases, the family GH23 enzymes have only a single identified catalytic residue at their catalytic centre. The identity of the catalytic acid/base residue of the lysozymes was first inferred by X-ray crystallography of goose egg-white lysozyme (GEWL) as Glu 73[5,6]. Likewise, analysis of the crystal structure of the soluble lytic transglycosylase 70 (Slt70) from Escherichia coli identified Glu as the lone catalytic residue[7]. Indeed, replacement of each respective residue results in loss of catalytic activity [8]. The mechanism of action of family GH23 enzymes has yet to be proven experimentally but examination of crystal structures and theoretical considerations has led to separate proposals for the two classes of enzymes. Based on the complexes formed with 1,6-anhydromuropeptide [9] or bulgecin [10], a substrate-assisted mechanism, analogous to the family GH18 chitinases and chitobiases and family GH20 N-acetyl-β-hexosaminidases, has been invoked for the lytic transglycosyales. Thus, the catalytic Glu73 is proposed to serve initially as an acid catalyst to donate a proton to the glycosidic oxygen of the linkage to be cleaved leading to the formation of an intermediate with oxocarbenium ion character (Figure 2). In the absence of an anion/nucleophile in close proximity to stabilize this oxocarbenium intermediate, the lytic transglycosylases would employ anchimeric assistance of the MurNAc 2-acetamido group resulting in the formation an oxazolinium ion intermediate. This would be followed by abstraction of the C-6 hydroxyl proton of the oxazolinium species involving Glu73 which now serves as the base catalyst leading to nucleophilic attack and the formation of 1,6-anhydromuramic acid product.

Three-dimensional structures

Three-dimensional structures are available for several Family GH23 enzymes, the first solved being that of GEWL[6]. The catalytic domain of each enzyme possesses the well characterized α+β “lysozyme fold” for avian lysozymes. However, there are distinct structural differences between the two classes of enzymes. Most notably, the environment of the active site in lytic transglycosylases, particularly around the catalytic acid/base, is more hydrophobic compared to that of GEWL. This distinction may account for the difference in the reaction mechanisms of the two enzymes.

Family Firsts

First identification of lytic transglycosylase

Soluble lytic transglycosylase 70 [1].

First catalytic nucleophile identification

Not applicable.

First general acid/base residue identification

Inferred by X-ray crystallography of goose egg-white lysozyme [2].

First 3-D structure

Goose egg-white lysozyme [3].

References

This is an example of how to make references to a journal article [4]. (See the References section below). Multiple references can go in the same place like this [4, 5]. You can even cite books using just the ISBN [1]. References that are not in PubMed can be typed in by hand [6].

1. Höltje JV, Mirelman D, Sharon N, and Schwarz U. (1975). Novel type of murein transglycosylase in Escherichia coli. J Bacteriol. 1975;124(3):1067-76. DOI:10.1128/jb.124.3.1067-1076.1975 |
3. Grütter MG, Weaver LH, and Matthews BW. (1983). Goose lysozyme structure: an evolutionary link between hen and bacteriophage lysozymes?. Nature. 1983;303(5920):828-31. DOI:10.1038/303828a0 |
7. Helland R, Larsen RL, Finstad S, Kyomuhendo P, and Larsen AN. (2009). Crystal structures of g-type lysozyme from Atlantic cod shed new light on substrate binding and the catalytic mechanism. Cell Mol Life Sci. 2009;66(15):2585-98. DOI:10.1007/s00018-009-0063-x |
8. Hirakawa H, Ochi A, Kawahara Y, Kawamura S, Torikata T, and Kuhara S. (2008). Catalytic reaction mechanism of goose egg-white lysozyme by molecular modelling of enzyme-substrate complex. J Biochem. 2008;144(6):753-61. DOI:10.1093/jb/mvn133 |
9. Romeis T, Vollmer W, and Höltje JV. (1993). Characterization of three different lytic transglycosylases in Escherichia coli. FEMS Microbiol Lett. 1993;111(2-3):141-6. DOI:10.1111/j.1574-6968.1993.tb06376.x |